Non-antigenic amine derived polymers and polymer conjugates

ABSTRACT

Substantially non-antigenic polymers containing pI and/or pH optimum modulating moieties are disclosed. The polymers are useful as intermediates for synthesis of amine-based polymers and in the formation of activated polymers for conjugation with nucleophiles. Conjugates and methods of preparation and treatment with the conjugates are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/265,593 filed Jun. 24, 1994 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polymers which are useful in extendingthe in vivo circulating life of biologically active materials. Theinvention also relates to conjugates made with the polymers.

2. Description of Prior Art

Some of the initial concepts of coupling peptides or polypeptides topoly(ethylene glycol) PEG and similar water-soluble poly(alkyleneoxides) are disclosed in U.S. Pat. No. 4,179,337, the disclosure ofwhich is incorporated herein by reference. Polypeptides modified withthese polymers exhibit reduced immunogenicity or antigenicity andcirculate in the bloodstream longer than unmodified versions.

To conjugate poly(alkylene oxides), one of the hydroxyl end-groups isconverted into a reactive functional group. This process is frequentlyreferred to as "activation" and the product is called an "activatedpoly(alkylene oxide)". Other substantially non-antigenic polymers aresimilarly "activated" or functionalized.

The activated polymers are reacted with a therapeutic agent havingnucleophilic functional group(s) that serve as attachment site(s). Onenucleophilic functional group commonly used as an attachment site is theepsilon-amino group of lysines. Free carboxylic acid groups, suitablyactivated carbonyl groups, oxidized carbohydrate moieties, hydroxyl andmercapto groups have also been used as attachment sites.

Over the years, one shortcoming observed with polymer conjugation isthat the conjugate has a different pI than the unmodified protein,enzyme, etc. For example, pegylation, i.e. attachment of the polymer, oflysine amino groups result in a decrease in the isoelectric point andchanges the pH optimum, i.e the pH at which maximum bioactivity isobserved. In some cases, it would be beneficial to restore the originalpI or even alter the pI value of the polymer conjugate in order tooptimize bioactivity at physiologic pH.

The present invention provides solutions to this problem.

SUMMARY OF THE INVENTION

In one aspect of the invention, there are provided amine-based polymerintermediates having the formula:

    R--B                                                       (I)

wherein (R) is a water-soluble, substantially non-antigenic polymerresidue and (B) is preferably a secondary amine or a tertiary amine.Suitable secondary amines are of the formula:

    --NH(CH.sub.2).sub.n X                                     (II)

wherein:

(n) is an integer from 1-12, inclusive; and

X is selected from the group consisting of H; OH; CO₂ R₁, where R₁ is Hor a C₁₋₆ alkyl; halogen, aryl moieties and heteroaromatic moieties.

Suitable tertiary amines can be selected from: ##STR1## wherein (m) isan integer from 0-12, inclusive;

(X) is selected from the group consisting of H, OH, CO₂ R₁, where R₁ isH or a C₁₋₆ alkyl, halogen, aryl moieties and heteroaromatic moieties;

(Y) is N or CH; and

(Z) is a C₁₋₆ alkyl.

The polymer is preferably a poly(alkylene oxide) residue such as apoly(ethylene glycol) having a molecular weight between about 200 andabout 100,000. In addition, the polymer is preferably capped with a C₁₋₄alkyl group on the end opposite (i.e. distal to) the B moiety.

In another aspect of the invention, there are provided substantiallynon-antigenic polymers of the formula:

    R--B'--W--L                                                (VII)

wherein (R) is as defined above; (B') is NY, where Y is H, i.e. asecondary amine or a C₁₋₆ alkyl or cycloalkyl; i.e. a tertiary amine (W)is a spacer group, such as a C₁₋₆ alkyl and (L) is a functional groupcapable of bonding with nucleophiles.

Other aspects of the invention include polymer conjugates containingbiologically-active materials, described as nucleophiles herein as wellas methods of conjugate preparation. In these aspects of the invention,the conjugates are of the formula:

     R--B'--W--L.sup.1 !.sub.z -(nucleophile)

wherein:

(R), (B') and (W) are as described above;

(L¹) represents a covalent linkage between (R--B'--W) and a nucleophile;and

(z) represents the number of polymers attached to the nucleophile.

The biologically active materials include proteins, peptides, enzymes,medicinal chemicals or organic moieties whether synthesized or isolatedfrom nature. The conjugation methods include contacting a nucleophile,i.e. a biologically active material, capable of undergoing asubstitution reaction with a polymer described. The reaction is carriedout under conditions sufficient to effect attachment while maintainingat least a portion of the biological activity.

The present invention also includes methods of treating various maladiesand conditions. In this aspect, a mammal in need of treatment isadministered an effective amount of a conjugate containing abiologically-active material such as a protein, enzyme or organic moietyattached to a polymer of the present invention.

Some of the chief advantages of the present invention are that theamine-derived polymers impart a pI and/or pH modulating effect to theconjugate. Thus, the isoelectric point of bio-active materials includedin the conjugate can be adjusted to a desired point. This is to becontrasted with the effect observed with conventional activated polymerswhere shifts in isoelectric points can be observed, often to thedetriment of optimal activity.

In addition, in those aspects of the invention where anionic groups areincluded as part of the amine-bearing moiety, the in vivo circulatinglife of the conjugates is further prolonged due to reduced renalfiltration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of synthesis techniques carried outin accordance with the invention to prepare formula (I) R--B polymerintermediates and the compounds of Examples 1 and 3-6.

FIG. 2 is a schematic representation of synthesis techniques carried outin accordance with the invention to prepare the compounds described inExamples 2 and 7-9.

FIG. 3 is a schematic representation of the reactions carried out inExamples 10-11.

FIG. 4 is a schematic representation of the reactions carried out inExamples 12-16.

DETAILED DESCRIPTION OF THE INVENTION

1. POLYMER RESIDUES

The activated polymers of the present invention are preferably preparedfrom poly(alkylene oxides) (PAO's) that are water soluble at roomtemperatures. Within this group are mono-substituted polyalkylene oxidederivatives such as methoxypoly (ethylene glycols) (mPEG's) or othersuitable alkyl-substituted PAO derivatives such as those containing a C₁-C₄ terminal group. Straight-chained non-antigenic polymers such asmonomethyl PEG homopolymers are preferred. Alternative polyalkyleneoxides such as other poly(ethylene glycol) homopolymers, polypropylenegylcols, other alkyl-poly(ethylene oxide) block copolymers, andcopolymers of block copolymers of poly(alkylene oxides) are also useful.Branched PAO's such as those disclosed in commonly-assigned the PCTInternational Application having International Publication No.WO95/11924 are also of use herein. The disclosure of WO95/11924 ishereby incorporated by reference herein.

Alternative polymer substances useful herein include materials such asdextrans, polyvinyl pyrrolidones, polyacrylamides or other similarnon-immunogenic polymers. Those of ordinary skill in the art willappreciate that these polymers can also be functionalized and includedin the invention. The foregoing is merely illustrative and not intendedto restrict the type of nonoantigenic polymers suitable for use herein.

Thus, (R) is a water-soluble, substantially non-antigenic polymerresidue. When the (R) is PEG or mPEG, molecular weights &between about200 and about 100,000 daltons are preferred; molecular weights of about1,000-50,000 daltons are more preferred and molecular weights of about2,500-25,000 daltons are most preferred.

2. AMINE PORTION OF ACTIVATED POLYMER

In one aspect of the invention, amine-based polymer intermediates areprovided. The amine portion of the intermediates can be a secondary,tertiary or quaternary amine moiety, represented herein as (B) informula (I). Preferably, however, either secondary or tertiary aminesare used.

When a secondary amine is selected for (B), amines of the formula (II)are preferred:

    --NH(CH.sub.2).sub.n X                                     (II)

wherein

(n) is an integer from 1-12, inclusive; and

(X) is selected from the group consisting of H; OH; CO₂ R₁, where R₁ isH or a C₁₋₆ alkyl; halogen; aryl moieties and heteroaromatic moieties.

These secondary amine moieties can be synthesized using standard organictechniques or can be readily obtained using reagents available fromcommercial sources. A non-limiting list of secondary amines meetingthese criteria include: primary amines such as ethanolamine,beta-alanine ethyl ester, paramethoxybenzylamine, etc.

When a tertiary amine is desired for (B), amines of formulae (III-VI)are preferred: ##STR2## wherein (m) is an integer from 0-12, inclusive;

(X) is selected from the group consisting of H; OH; CO₂ R₁, where R₁ isH or a C₁₋₆ alkyl; halogen, aryl moieties and heteroaromatic moieties;

(Y) is N or CH; and

(Z) is a C₁₋₆ alkyl.

The tertiary amine moieties can be synthesized using standard organictechniques or can be readily obtained from commercial sources. Anon-limiting list of tertiary amines meeting these criteria includesecondary amines such as N-methylethanolamine, piperidine-3-methanol,N-hydroxypropylpiperazine, sarcosine ethyl ester, etc.

3. SYNTHESIS OF POLYMERS INTERMEDIATES

The polymer intermediates of formula (I) can be synthesized usingconventional reaction techniques. For example, mPEG-Cl can be reactedwith ethanolamine available from Aldrich or any other suitable primaryamine in water and a sealed container at temperatures of approximately60° C. to form secondary amine polymer intermediates.

Similarly, mPEG-Cl can be reacted with secondary amines such asN-methylethanolamine under similar conditions to form tertiary aminepolymer intermediates.

In still other aspects of the invention, the amine-bearing groups (B)can also include an acid moiety, preferably as part of the alkyl portionthereof. A non-limiting list of suitable acids include sulfonates,carboxylates and phosphonates. As demonstrated in Examples 12-16, atrifunctional spacer moiety such as lysine or serine is first reactedwith an activated polymer such as a PEG-succinimidyl succinate ester ora PEG-succinimidyl carbonate. Next, an anion group is added by modifyingone of the remaining functional groups on the (B) portion of theintermediate. Reagents such as chloroethane sulfuric acid sodium salt,phosphoric acid esters or salts or ethylbromoacetate can be used.(Ethylbromoacetate first forms a carboxylate ester and then can besaponified to a salt.) Finally, the third functional group of (B) can bemodified with a leaving group (L) described below in section 4.

The foregoing is merely illustrative of syntheses useful to prepare theintermediates. It is to be understood that alternative methods andreagents may be used without departing from the spirit of the invention.In essence, each (R) and (B) is joined by a reaction betweennucleophilic functional groups. Each (R) is suitably functionalized,i.e. mPEG-halides such as mPEG-Cl; PEG sulfonate esters such asPEG-tosylate, etc. to undergo nucleophilic substitution and bond with(B). Functionalization of polymers with halides or sulfonate esters isreadily apparent to those of ordinary skill in the art.

4. Activated Amine-Based Polymers

In yet another aspect of the invention, the intermediates describedabove are included as part of activated polymers which can be used toconjugate with biologically-active nucleophiles. The polymers have theformula:

    R--B'--W--L                                                (VII)

wherein

(R) is a water-soluble, substantially non-antigenic polymer residue;

(B') is NY, wherein Y is H, i.e. a secondary amine or a C₁₋₆ alkyl orcycloalkyl, i.e. a tertiary amine;

(W) is a spacer group; and

(L) is a functional group capable of bonding with nucleophiles.

The (R--B') portion of the polymer can be prepared as described abovewith regard to (R--B).

The moiety (W) of Formula (VII) represents a spacer group between the(R--B') and the leaving group (L). It has been found that straight orbranched lower alkyls i.e. C₁₋₄, aralkyl, aryl or heteroaryl groups maybe employed. In this regard, alternative (W) substitutents includebenzyl, substituted benzyls, phenylethyl, substituted phenylethyls,phenyl, substituted phenyls, thiophene derivatives, pyridinederivatives, hydroxyanaline, etc. The foregoing list of (W)substitutents is to understood as being illustrative of suitablemoieties and not an exclusive list. Those of ordinary skill will realizethat functional equivalents of the foregoing are also possible for (W).

The spacer adjusts the geometry of the system in order to preventundesirable side reactions, thus increasing the yield of the desiredpolymer-nucleophile conjugate.

Preferred spacer groups (W) include:

1. ethyl-3-isocyanato propionate; and

2. ethyl-2-aminobutyrate.

The spacer groups are available from commercial sources or can besynthesized using standard laboratory techniques.

Furthermore, the spacer groups can be attached to the (R--B) portion ofthe polymer by simple nucleophilic or electrophilic substitutionreactions and as a result, modifying the (X) portion of the secondary ortertiary amines of Formulas (II)-(VII).

The moiety (L) of Formula (VII) represents leaving groups that"activate" the amine-based polymers of the present invention forconjugation with nucleophiles.

(L) can be a moiety selected from:

I. Functional groups capable of reacting with an amino group such as:

a) carbonates such as the p-nitrophenyl, or succinimidyl;

b) carbonyl imidazole;

c) azlactones;

d) cyclic imide thiones;

e) isocyanates or isothiocyanates; or

f) other active esters.

II. Functional groups capable of reacting with carboxylic acid groupsand reactive carbonyl groups such as:

a) primary amines; or

b) hydrazine and hydrazide functional groups such as acyl hydrazides,carbazates, semicarbazates, thiocarbazates, etc.

III. Functional groups capable of reacting with mercapto groups such asunsaturated maleimides.

5. Synthesis pf Activated Amine-Based Polymers

The activated polymers are formed using conventional reactiontechniques. For example, an alcohol form of the intermediate (R--B),formula (I), or the intermediate (R--B'--W) of formula (VII) can bereacted with N-hydroxysuccinimide and diisopropylcarbodiimide, bothavailable from Aldrich, in dry methylene chloride at a temperature ofabout 25° C. Other inert organic solvents such as toluene, THF, etc canbe used and temperatures ranging from 1°-100° C. can be employed.Synthesis reactions are schematically shown in FIGS. 1-4 for bothsecondary and tertiary amine-containing activated polymers.

6. Biologically Active Materials for Conjugation

The nucleophiles conjugated with the polymers are described as"biologically active". The term, however, is not limited tophysiological or pharmacological activities. For example, somenucleophile conjugates such as those containing enzymes, are able tocatalyze reactions in organic solvents. Likewise, some inventive polymerconjugates containing proteins such as concanavalin A, immunoglobulinand the like are also useful as laboratory diagnostics. A key feature ofall of the conjugates is that at least some portion of the activityassociated with the unmodified bio-active material is maintained.

In other aspects of the invention, the conjugates are biologicallyactive and have numerous therapeutic applications. Mammals in need oftreatment which includes a biologically active material can be treatedby administering an effective amount of a polymer conjugate containingthe desired bioactive material. For example, mammals in need of enzymereplacement therapy or blood factors can be given polymer conjugatescontaining the desired material. The therapeutically effective doses ofthe conjugates will be apparent to those of ordinary skill in theinvention. It is contemplated, however, that the doses will becalculated of the basis of the biologically active material present inthe conjugates.

Biologically active nucleophiles of interest of the present inventioninclude, but are not limited to, proteins, peptides, polypeptides,enzymes, organic molecules of natural and synthetic origin such asmedicinal chemicals and the like.

Enzymes of interest include carbohydrate-specific enzymes, proteolyticenzymes, oxidoreductases, transferases, hydrolases, lyases, isomerasesand ligases. Without being limited to particular enzymes, examples ofenzymes of interest include asparaginase, arginase, arginine deaminase,adenosine deaminase, superoxide dismutase, endotoxinases, catalases,chymotrypsin, lipases, uricases, adenosine diphosphatase, tyrosinasesand bilirubin oxidase. Carbohydrate-specific enzymes of interest includeglucose oxidases, glucodases, galactosidases, glucocerebrosidases,glucouronidases, etc.

Proteins, polypeptides and peptides of interest include, but are notlimited to, hemoglobin, serum proteins such as blood factors includingFactors VII, VIII, and IX; immunoglobulins, cytokines such asinterleukins, α-, β- and γ-interferons, colony stimulating factorsincluding granulocyte colony stimulating factors, platelet derivedgrowth factors and phospholipase-activating protein (PLAP). Otherproteins of general biological or therapeutic interest include insulin,plant proteins such as lectins and ricins, minor necrosis factors andrelated aleles, growth factors such as tissue growth factors, such asTGFα's or TGFβ's and epidermal growth factors, hormones, somatomedins,erythropoietin, pigmentary hormones, hypothalamic releasing factors,antidiuretic hormones, prolactin, chorionic gonadotropin,follicle-stimulating hormone, thyroid-stimulating hormone, tissueplasminogen activator, and the like. Immunoglobulins of interest includeIgG, IgE, IgM, IgA, IgD and fragments thereof.

Some proteins such as the interleukins, interferons and colonystimulating factors also exist in non-glycosylated form, usually as aresult of using recombinant techniques. The non-glycosylated versionsare also among the biologically active nucleophiles of the presentinvention.

The biologically active nucleophiles of the present invention alsoinclude any portion of a polypeptide demonstrating in vivo bioactivity.This includes amino acid sequences, antisense moieties and the like,antibody fragments, single chain binding antigens, see, for example U.S.Pat. No. 4,946,778, disclosure of which is incorporated herein byreference, binding molecules including fusions of antibodies orfragments, polyclonal antibodies, monoclonal antibodies, catalyticantibodies, nucleotides and oligonucleotides.

The proteins or portions thereof can be prepared or isolated by usingtechniques known to those of ordinary skill in the art such as tissueculture, extraction from animal sources, or by recombinant DNAmethodologies. Transgenic sources of the proteins, polypeptides, aminoacid sequences and the like are also contemplated. Such materials areobtained form transgenic animals, i.e., mice, pigs, cows, etc., whereinthe proteins expressed in milk, blood or tissues. Transgenic insects andbaculovirus expression systems are also contemplated as sources.Moreover, mutant versions of proteins, such as mutant TNF's and/ormutant interferons are also within the scope of the invention.

Other proteins of interest are allergen proteins such as ragweed,Antigen E, honeybee venom, mite allergen, and the like. However, usefulbiologically active nucleophiles are not limited to proteins andpeptides. Essentially any biologically-active compound is includedwithin the scope of the present invention. Chemotherapeutic moleculessuch as pharmaceutical chemicals i.e. anti-tumor agents such as taxol,taxanes, camptothecin, anthracyclines, methotrexate, etc.,cardiovascular agents, anti-neoplastics, anti-infectives, anti-anxietyagents, gastrointestinal agents, central nervous system-activatingagents, analgesics, fertility or contraceptive agents, anti-inflammatoryagents, steroidal agents, anti-uricemic agents, cardiovascular agents,vasodilating agents, vasoconstricting agents and the like can all beconjugated with the amine-bearing polymers of the present invention.

The foregoing is illustrative of the biologically active nucleophileswhich are suitable for conjugation with the polymers of the invention.It is to be understood that those biologically active materials notspecifically mentioned but having suitable nucleophilic groups are alsointended and are within the scope of the present invention.

7. Synthesis of Biologically Active Conjugation

One or more of the activated polymers can be attached to a biologicallyactive nucleophile by standard chemical reactions. The conjugate isrepresented by the formula:

     R--B'--W--L.sup.1 !.sub.z -(nucleophile)                  (VIII)

wherein:

(R) is a water-soluble substantially non-antigenic polymer residue;

(B') is NY, wherein Y is H, i.e. a secondary amine or a C₁₋₆ alkyl orcycloalkyl, i.e. a tertiary amine;

(W) is a spacer moiety;

(L¹) represents a covalent linkage between (R--B'--W) and thenucleophile; and

(z) Represents the number of polymers attached to the nucleophile.

The variable (z) is an integer≧1 representing the number of polymersconjugated to the biologically active nucleophile. The upper limit for(z) will be determined by the number of available nucleophilicattachment sites and the degree of polymer attachment sought by theartisan. The degree of conjugation can be modified by varying thereaction stoichiometry using well-known techniques. More than onepolymer conjugated to the nucleophile can be obtained by reacting astoichiometric excess of the activated polymer with the nucleophile.

The biologically active nucleophiles can be reacted with the activatedpolymers in an aqueous reaction medium which can be buffered, dependingupon the pH requirements of the nucleophile. The optimum pH for thereaction is generally between about 6.5 and about 8.0 and preferablyabout 7.4 for proteinaceous/polypeptide materials.Organic/chemotherapeutic moieties can be reacted in non-aqueous systems.The optimum reaction conditions for the nucleophile's stability,reaction efficiency, etc. is within level of ordinary skill in the art.The preferred temperature range is between 4° C. and 37° C. Thetemperature of the reaction medium cannot exceed the temperature atwhich the nucleophile may denature or decompose. It is preferred thatthe nucleophile be reacted with an excess of the activated polymer.Following the reaction, the conjugate is recovered and purified such asby diafiltration, column chromatography, combinations thereof, or thelike.

It can be readily appreciated that the activated non-antigenic polymersof the present invention are a new and useful tool in the conjugation ofbiologically active materials, especially when shifts in pI and/or pHoptimum are sought.

EXAMPLES

The following non-limiting examples illustrate certain aspects of theinvention. All parts and percentages are by weight unless otherwisenoted and all temperatures are in degrees Celsius. The numbersassociated with all compositions in the examples correspond to thoseshown in the figures.

MATERIALS

Methoxypoly(ethylene glycol) (m-PEG) having a molecular weight of about5,000 was obtained from Union Carbide. All polyethylene glycols weremolecular weight 5,000 unless stated otherwise. The solvents wereobtained from Aldrich Chemical of Milwaukee, Wis. Each of the productsprepared was confirmed structurally by carbon--13 NMR.

Example 1

m-PEG-Cl

A total of 1 kg (0.2 moles) of m-PEG-OH was placed under high vacuum andheated to 70° C. with stirring until the evolution of gas (H₂ O) ceasedafter approximately 4 hours. The temperature was lowered to 50° C.,followed by the addition of 100 ml (1.4 moles) of thionyl chloride. Thismixture was stirred overnight at 70° C. under nitrogen, cooled to 30°C., and titrated with ethyl ether to yield 990 g (99% yield) of product.¹³ C NMR: (CDCl₃)δ; CH₂ Cl, 42.3.

Example 2

m-PEG Piperidinemethanol (7)

A mixture of 25.0 g (5.0 mmoles) of m-PEG-Cl and 5.75 g (50 mmoles) of3-piperidinemethanol (6) in 100 ml of water was placed in a 250 mlpolypropylene bottle. The bottle was sealed and kept in a water bath at60° C. for 48 hours followed by cooling to room temperature and removalof the solvent by distillation in vacuo. Recrystallization of theresidue from 2-propanol yielded of product (7). ¹³ C NMR: (CDCl₃ δ); CH₂(ring), 23.94, 26.47; CHCH₂ OH, 37.56; CH₂ N, 53.91, 56.96, 57.38; CH₃O, 57.93; CH₂ OH, 65.05.

Example 3

m-PEG Piperazinepropanol (2)

A mixture of 25.0 g (5.0 mmoles) of m-PEG-Cl and 7.2 g (50 mmoles) ofN-hydroxypropylpiperazine (1) in 100 ml of water was placed in a 250 mlpolypropylene bottle. The bottle was sealed and kept in a water bath at60° C. for 48 hours followed by cooling to room temperature and removalof the solvent by distillation in vacuo. Recrystallization of theresidue from 2-propanol yielded 24.5 g (96% yield) of product (2). ¹³ CNMR: (CDCl₃)δ; CH₂ CH₂ OH, 26.85; CH₂ N (endocyclic), 52.19, 52.48; CH₂N (exocyclic), 56.66, 56.81; CH₃ O, 57.90; CH₂ OH, 62.18.

Example 4 Synthesis of Compound (3a)

mPEG-piperazine propanol (2), (16 g, 3.1 mmol) in toluene (240 ml) wasazeotroped for 2 hours, followed by the removal of 100 ml toluene.Thereafter, ethyl-3-isocynatopropionate (1.34 g, 9.36 mmol) and dibutyltin dilaurate (0.99 g, 1.56 mmol) were added to this solution at roomtemperature. The resulting mixture was heated to 60° C. for 30 hours.The solvent was removed under reduced pressure and the solid obtainedwas recrystallized from 2-propanol to give (3a) in 96% yield. ¹³ C NMR:(CDCl₃) δ; 13.43 (OCH₂ CH₃); 25.40 (CH₂ CH₂ CH₂); 33.63 (CH₂ C); 35.73(CNCH₂); 51.56, 52.19 (CH₂ N endocyclic); 53.92, 56.61 (CH₂ Nexocyclic); 58.11 (OCH₃); 59-60 (OCH₂ CH₃); 62.01 (CH₂ OC); 155.62(OCN); 171.14 (COC₂ H₅).

Example 5 Synthesis of Compound (3b)

Compound (3a) (15 g, 2.9 mmol) was dissolved in H₂ O (200 mol) followedby the addition of lithium hydroxide (733 mg, 125 mmol). The solutionwas stirred for 18 hours and pH was adjusted to 4.0 using 6N HCl. Theaqueous solution was extracted with methylene chloride and the solventwas removed in vacuo. The solid obtained was recrystallized from CH₂ Cl₂/ether to give (3b) in 96.6% yield. ¹³ C NMR: (CDCl₃) δ; 24.21 (CH₂ CH₂CH₂); 33.49 (CH₂ C); 35.94 (NHCH₂); 49.71, 50.52 (CH₂ N endocyclic);53.29, 55.79 (CH₂ N exocyclic); 58.12 (OCH₃); 61.19 (CH₂ OC); 155.49(OCN); 172.96 (COH).

Example 6 A. Synthesis of Compound (4)

Compound 3b (14 g, 2.71 mmol) was dissolved in anhydrous methylenechloride (70 ml) followed by the addition of N-hydroxysuccinimide (619mg; 5.4 mmol) and 1,3-diisopropyl carbodiimide (683 mg, 5.42 mmol). Themixture was stirred overnight at room temperature. The resulting solidswere filtered and the solvent was removed under reduced pressure. Thecrude product was recrystallized from 2-propanol to give compound (4) in94% yield. ¹³ C NMR: (CDCl₃) δ; 24.84 (CH₂ CH₂ CH₂,CH₂, succinimide);30.94 (CH₂ C); 35.74 (NCH₂); 49.84, 49.98, 50.05, 50.74 (CH₂ Nendocyclic); 53.29, 55.90 (CH₂ N exocyclic); 58.15 (OCH₃); 61.48 (CH₂OC); 155.56 (CN); 166.51 (CH₂ CO); 168.73 (C succinimide).

B. Synthesis of Compound (5) by Conjugation of Compound (4) withBenzylamine

Benzylamine (9 μl) was added to the solution of compound (4) (0.4 g) inCDCl₃ in the NMR tube. Resulting solids were dissolved by gentle heatingand tapping and left at room temperature for 18 hours. ¹³ C NMR of theresulting solution indicates the formation of product. Thus, it is shownthat the amine-based polymers of the present invention react readilywith nucleophiles to form conjugates. ¹³ C NMR: (CDCl₃) δ; 24.67 (CH₂CH₂ CH₂); 24.58 (CH₂ free succinimide); 34.87 (CH₂ C); 36.50 (HNCH₂);42.31, 42.81 (CH₂ benzyl); 50.76, 51.18 (CH₂ N endocyclic); 53.58, 56.16(CH₂ N); 58.12 (CH₃ O); 61.50 (CH₂ OC); 126.77 to 138.11 (C Benzenering); 155.67 (CN); 170.77 (CH₂ CON); 172.55 (CO free succinimide).

Example 7 Synthesis of Compound (8a)

mPEG-piperidine methanol (7) (5 g, 1 mmol) in toluene (75 ml) wasazeotroped for 2 hours, followed by the removal of 30 ml toluene. Tothis solution ethyl-3-isocyanate propionate (421 mg, 2.9 mmol) anddibutyl tin dilaurate (310 mg, 0.49 mmol) were added at room temperaturewere added and the resulting mixture was heated to 60° C. for 30 hours.The solvent was removed under reduced pressure and the solid obtainedwas recrystallized from 2-propanol to give (8a) in 91% yield. ¹³ C NMR:(CDCl₃) δ; 13.30 (OCH₂ CH₃); 23.32 (CH₂ ring); 33.47 (CH₂ C); 35.65(NCH₂); 34.75 (CH₂ ring); 53.37, 56.32 (CH₂ N); 57.94, (OCH₃); 59.42(OCH₂ CH₃); 66.17 (CH₂ OC); 155.49 (CN); 170.92 (CH₂ CO).

Example 8 Synthesis of Compound (8b)

Compound (8a), (4.5 g 0.86 mmol) was dissolved ) in H₂ O (60 ml)followed by the addition of lithium hydroxide (216 mg, 5.15 mmol). Thesolution was stirred for 18 hours and pH was adjusted to 4.0 using 6NHCl. The aqueous solution was extracted with CH₂ Cl₂ and the solvent wasremoved under reduced pressure. The solid obtained was recrystallizedfrom CH₂ Cl₂ /ether mixture to give (8b) in 98% yield. ¹³ C NMR: (CDCl₃)δ; 21.23, 24.19 (CH₂ ring); 32.95 (CH₂ C); 133.88 (CH ring); 36.12(NCH₂); 57.99, (OCH₃); 64.90 (CH₂ OC); 155.27 (OCN); 173.56 (C--OH).

Example 9 A. Synthesis of Compound (9)

Compound (8b) (2 g, 0.38 mmol) was dissolved in anhydrous methylenechloride (10 ml) followed by the addition of N-hydroxysuccinimide (88.3mg, 0.77 mmol) and 1,3diisopropylcarbodiimide (96.7 mg, 0.77 mmol). Themixture was stirred overnight at room temperature. The resulting solidswere filtered and the solvent was removed under reduced pressure. Thecrude product was recrystallized from 2-propanol to give compound (9) in88% yield. ¹³ C NMR: (CDCl₃) δ; 21.67, 24.65 (CH₂ ring); 24.50 (CH₂succinimide); 30.62 (CH₂ C); 33.36 (CH ring); 35.58 (NCH₂); 52.64,54.98, 55.06 (CH₂ N); 57.94 (OCH₃); 155.32 (OCN); 166.31 (CH₂ CO);168.60 (succinimide).

B. Synthesis of Compound (10) by Conjugation with Compound (9) withBenzylamine

Benzylamine (9.1 μl) was added to the solution of compound (9) (0.4 g)in CDCl₃ in the NMR tube. Resulting solids were dissolved by gentleheating and tapping and left at room temperature for 18 hours. ¹³ C NMRof the resulting solution indicates the formation of product (10). ¹³ CNMR: (CDCl₃) δ; 21.80, 21.49 (CH₂ ring); 24.39 (CH₂, succinimide); 33.44(CH₂ C); 36.39 (CH₂ N); 34.72 (CH ring); 42.08, 42.65 (CH₂ benzylamine);52.51, 55.07, 55.98 (CH₂ N); 57.91 (OCH₃); 126.06 to 138.03 (C Benzenering); 155.53 (OCN); 170.56 (CH₂ CN); 172.31 (C succinimide).

Example 10 Synthesis of N-Peg Sarcosine (12)

mPEG-Cl (25 g, 4.98 mmol) was added to the solution of N-methyl glycine(sarcosine) (11) in NaOH solution (150 ml, 0.33M) and the mixture wasplaced in a sealed polypropylene bottle and heated at 75° C. for 4 days.The reaction mixture was cooled to room temperature and pH was adjustedto 6.0/6.5 with dilute HCl. The aqueous solution was extracted with CH₂Cl₂ and the solvent was removed under reduced pressure. The resultingsolid was recrystallized from 2-propanol to give compound (12) in 77%yield. ¹³ C NMR: (CDCl₃) δ; 41.31 (NCH₃); 54.57 (CH₂ N); 57.85 (CH₂ C);58.04 (OCH₃); 167.98 (CO).

Example 11 A. Synthesis of Compound (13)

mPEG sarcosine (12) (18 g, 3.55 mmol) was dissolved in anhydrousmethylene chloride (90 ml) followed by the addition ofN-hydroxysuccinimide (612 mg, 5.32 mmol) and diisopropyl carbodiimide(671 mg, 5.32 mmol). The mixture was stirred overnight at roomtemperature. The resulting solids were filtered and the solvent wasremoved in vacuo. The crude product was recrystallized from 2-propanolto give the compound (13) in 94% yield. ¹³ C NMR: (CDCl₃) δ; 24.73 (CH₂succinimide); 41.60 (N--CH₃); 54.57 (NCH₂); 54.44 (CH₂ C); 58.11 (OCH₃);165.13 (CO) 168.48 (C succinimide).

B. Synthesis of Compound (14) by Conjugation with Compound (13) withBenzylamine

Benzylamine (41.45 mg, 0.39 mmol) was added to Compound (13) (1 g, 0.19mmol) in anhydrous CH₂ Cl₂ (5 ml). The reaction mixture was kept at roomtemperature overnight. The solvent was removed under reduced pressureand the residue was recrystallized from 2-propanol to give compound (14)in 80% yield. ¹³ C NMR: (CDCl₃) δ; 41.94 (NCH₃); 42.65 (CH₂Benzylamine); 56.32 (NCH₂); 58.12 (OCH₃); 60.22 (CH₂ C); 126.32 to137.99 (C Benzene ring);169.70 (CO).

Example 12

N-epsilon-Boc lysine (15), (0.246 g, 1 mmol) PEG5,000-N-hydroxysuccinimide ester (16) (5 g, 1 mmol) are added to pH 8borate buffer (30 ml). The mixture is stirred at room temperature forabout 3 hours and then neutralized with dilute hydrochloric acid. Theaqueous solution was extracted with dichloromethane (3×20 ml). Theorganic layer was dried (anhyd. Na₂ SO₄) and evaporated under reducedpressure to yield 4.1 g, 79% N-epsilon-tert.-butoxycarbonyl-alphaPEGcarbamate of L-lysine (17).

Example 13

N-epsilon-tert.-butoxycarbonyl-alpha PEGcarbamate of L-lysine (17) (5.2g; 1.0 mmol) is dissolved in trifluoroacetic acid and dichloromethane(1:2, 30 ml) and left to stand at room temperature for one hour. Thesolution is evaporated to dryness in vacuo and the residue is trituratedwith dry ether, filtered, washed with ether and dried in vacuo to yield(3 g, 60%) of lysine-N-alpha-PEGcarbamate (18).

Example 14

A mixture of lysine-N-alpha-PEGcarbamate (18) (5.1 g, 1 mmol)chloroethane sulfonic acid sodium salt (1.84 g, 10 mmol) in 20 ml ofwater is placed in a 10 ml polypropylene bottle. The bottle is sealedand kept in a water bath at 60° C. for 48 hours followed by cooling toroom temperature and removal of the solvent by distillation in vacuo.Recrystallization of the residue from 2-propanol yields (5.5 g, 80%) ofthe compound (19).

Example 15

A solution of the compound (19) (5.2 g, 1 mmol) and N-hydroxysuccinimide(115 mg, 1 mmol) in dry dichloromethane is cooled in an ice water bathand diisopropylcarbodiimide (0.126 g. 1 mmol) is added with stirring.The mixture is kept in a refrigerator overnight. The solvent is removedand the solid obtained is recrystallized from 2-propanol to give 4.3 g,(80%) of the product (19).

Example 16

The following procedure details the conjugation of bovine hemoglobin(Hb), 9.8% solution, using an 18 fold molar excess of piperidine pI PEG(9) in the presence of 10% NaCl.

Piperidine pI PEG (9) 1.461 g, 2.842×10⁻⁴ M, is dissolved in 2 ml of pH7.0 phosphate buffer (0.1M) in a jacketed reaction vessel (0° C.) andstirred for 10 minutes. Ten ml of the Hb solution is pipetted intoanother jacketed reaction vessel (8° C.), along with 2.3 g NaCl. The pHis adjusted to 7.8 The pI PEG solution is added to the reaction vesselcontaining the Hb and the container is washed with 2×0.5 ml of the pH7.0 buffer. The washings are then transferred to the Hb vessel. Seven mlof pH 7.8 phosphate buffer is added and the pH is adjusted to 7.8 with1.0N NaOH. The reaction mixture is stirred uniformly and slowly forabout 2 hours at 8° C. while maintaining the pH at 7.8.

After 2 hours, the reaction is quenched with cysteine HCl (0.105 g) and124 μl of glycine solution (2.25×10⁻⁴ M). The pH is adjusted to 7.8 with2N NaOH. The conjugate can now be stored in the refrigerator between4°-6° C.

As will be readily appreciated, numerous variations and combinations ofthe features set forth above can be utilized without departing from thepresent invention as set forth in the claims. Such variations are not tobe regarded as a departure from the spirit and scope of the invention,and all such modifications are intended to be included within the scopeof the following claims.

What is claimed is:
 1. A substantially non-antigenic polymer comprisingthe formula:

    R--B'--W--L

wherein (R) is a water-soluble, substantially non-antigenic polymerresidue; (B') is a secondary or tertiary amine when B' is a tertiaryamine, (W) is a spacer moiety selected from the group consisting ofstraight or branched C₁₋₄ alkyls, aralkyl, aryl and heteroaryl moieties,C(O)NHCH₂ CH₂ C(O) and CH₂ C(O); when B' is a secondary amine, (W) isselected from the group consisting of aralkyl, aryl, heteroarylmoieties, C(O)NHCH₂ CH₂ C(O) and CH₂ C(O); and (L) is selected from thegroup consisting of: OH; functional groups capable of reacting withamino groups; functional groups capable of reacting with carboxylic acidgroups; functional groups capable of reacting with reactive carbonylgroups; and functional groups capable of reacting with mercapto groups.2. The polymer of claim 1, wherein said secondary amine comprises theformula:

    HN--(CH.sub.2).sub.n X,

wherein: (n) is an integer from 1-12, inclusive; and (X) is selectedfrom the group consisting of H; OH; CO₂ R₁, where R₁ is H or a C₁₋₆alkyl; halogen; aryl moieties and heteroaromatic moieties.
 3. Thepolymer of claim 1, wherein said tertiary amine comprises a formulaselected from the group consisting of: ##STR3## wherein (m) is aninteger from 0-12 inclusive;(X) is selected from the group consisting ofH; OH; CO₂ R₁, where R₁ is H or a C₁₋₆ alkyl; halogen; aryl moieties andheteroaromatic moieties; (Y) is N or CH; and (Z) is a C₁₋₆ alkyl.
 4. Thepolymer of claim 1, wherein said polymer is a poly(alkylene oxide)residue.
 5. The polymer of claim 4, wherein said poly(alkylene oxide)residue is capped with a C₁₋₄ alkyl group distal to said (B') moiety. 6.The polymer of claim 4, wherein said poly(alkylene oxide) residue isselected from the group consisting of poly(ethylene glycol)homopolymers, alkyl-capped poly(ethylene oxides), and copolymers ofblock copolymers of poly(alkylene oxides).
 7. The polymer of claim 6,wherein said poly(alkylene oxide) has a molecular weight between about200 and about 100,000.
 8. The polymer of claim 7, wherein saidpoly(alkylene oxide) has a molecular weight between 1,000 and about50,000.
 9. The polymer of claim 8, wherein said poly(alkylene oxide) hasa molecular weight of about 2,500 to about 25,000.
 10. The polymer ofclaim 1, wherein said moiety (W) is selected from the group consistingof ethyl-3-isocyanato propionate and ethyl-2-aminobutyrate.
 11. Thepolymer of claim 1, wherein (L) is a succinimidyl or a p-nitrophenylcarbonate active ester.
 12. The polymer of claim 11, wherein saidfunctional group capable of reacting with an amino group is selectedfrom the group consisting of carbonyl imidazole, azlactones, cyclicimide thiones, isocyanates, isothiocyanates and active esters.
 13. Thepolymer of claim 11, wherein said functional group capable of reactingwith carboxylic acid groups is selected from the group consisting ofprimary amines, hydrazine, acyl hydrazides, carbazates, semicarbazatesand thiocarbazates.
 14. The polymer of claim 11, wherein said functionalgroup capable of reacting with reactive carbonyl groups is selected fromthe group consisting of primary amines, hydrazine, acyl hydrazides,carbazates, semicarbazates and thiocarbazates.
 15. The polymer of claim11, wherein said functional group capable of reacting with mercaptogroups is an unsaturated maleimide.
 16. The polymer of claim 14, whereinsaid poly(alkylene oxide) residue is a poly(ethylene glycol) residue.17. A polymer of claim 11 having a structure selected from the groupconsisting of: ##STR4##